Haplotype information can be vital in the analysis of disease by determining whether two or more sequence variants are located on the same nucleic acid fragment. This is of special interest in tumour research and diagnosis where it is important to know if two or more inactivating mutations occur on the same or different chromosomes. Similarly better information about which genotypes are located on the same nucleic acid segment can greatly increase the information derived from genotyping data and in the statistical analysis of genetic linkage or linkage disequilibrium of inherited traits and markers such as single nucleotide polymorphisms, SNPs (e.g. [1-3]).
To date there has been no method developed that satisfactorily solves the problem of how to obtain haplotype information in vitro, that is to determine which gene variants are located, over some distance, on the same nucleic acid molecule.
Current analysis of heritable diseases is hampered by the fact that given the genotypes of the parents, it is still often impossible to confirm if a particular allele is obtained from the mother or from the father. In theory, with the ability to distinguish between haplotypes, more meioses would be informative and thereby facilitate genetic linkage analysis. Another area where haplotyping has proven to be of interest is in the study of genetic effects on a subject's response to different drugs. Recent publications have shown that haplotype information is important to be able to relate genetic factors to a patient's response to various drugs, [4].
Currently, there are only a few methods available for obtaining haplotype information. When lineage data and nucleic acid samples are available linkage analysis is applicable. It is also possible to use statistical methods to calculate possible allele-combinations from allele frequencies to gain information on a haplotype. However, this technique can only be used with a small number of alleles at the same time and on population-size data, and the analysis only provides statistical evidence for the presence of a given haplotype. Haplotype information can also be gained from hemizygous X- and Y-chromosomes, where haplotypes are immediately apparent from the genotype.
The possibility to study cells with only one autosome chromosome is utilised in some in vivo techniques. One approach is the creation of rodent-human hybrid cells, for example using the so called “Conversion technology” [2]. Some of the rodent-human hybrid cells will contain one of the two possible copies from a human chromosome. A second approach is to use hydatidiform moles, i.e. tissues that due to a fertilisation defect only contain genetic material from the sperm (complete hydatidiform mole) thereby containing only one copy of each chromosome.
There are also some in vitro molecular techniques that can be used to determine haplotypes. One technique is the sub-cloning of all nucleic acid sequences of interest, isolating individual clones and subsequently genotyping them. Allele-specific analysis through Fibre Fluorescent In Situ Hybridisation is another possible approach, however it has not yet been convincingly shown to be useful for SNP based haplotyping. A third approach is double PCR Allele Specific Amplification (double-PASA [5], a double allele-specific Polymerase Chain Reaction (PCR) which gives linkage information of two adjacent polymorphic sites. Pyrosequencing [6] and mass spectrometry may be used to analyse haplotypes over short distances, i.e. <100 nt.